For in vitro Veterinary Diagnostics only. Real-Time PCR Detection Reagents for Detection of Marek Disease Virus and differentiation between field strains and Rispens vaccine CVI988 strain www.kylt.eu
DIRECTION FOR USE Art. No. 31418 / 31419 Kylt MDV & Rispens DIVA Real-Time PCR Detection Reagents for Detection of Marek Disease Virus and differentiation between field strains and Rispens vaccine strain CVI988 100 / 25 reactions in vitro Diagnosticum for birds A. General Kylt MDV & Rispens DIVA Real-Time PCR Detection Kit is for detection of Marek Disease Virus and differentiation of field strains and Rispens vaccine CVI988 strain in samples from birds, such as feathers, tissues and organs and sampling material derived from cultural processes with the aforementioned samples. Kylt MDV & Rispens DIVA comprises the Detection-Mix and controls for detection of viral DNA of Marek Disease virus. To conduct an analysis for MDV an additional, at least 2x concentrated qpcr-mix is required, please inquire for recommendations. The qualitative testing of Kylt MDV & Rispens DIVA is based on a quadruplex Real-Time PCR system: After DNA extraction the target gene specific for MDV as well as for field strains, the Rispens vaccine strain and the Internal Amplification Control are amplified in parallel by respective primer pairs in the subsequent Polymerase Chain Reaction (PCR) in one reaction vessel. Amplified target gene fragments are detected via fluorescently labeled probes during the PCR reaction in real-time (Real-Time PCR). The probes specific for detection of target genes of amplified MDV, MDV field strains, MDV Rispens vaccine strain and Internal Amplification Control are labeled with fluorescent dyes FAM, Texas Red, Cy5 and HEX, respectively, and their emitted fluorescence is separately optically measured by the Real-Time PCR thermal cycler. By means of four individual analyses in one reaction vessel per sample and the Negative Control and Positive Control per run the MDV-specific status of a sample can be evaluated in the end. This way, results can be achieved within few hours after sample receipt. This kit was developed for use by trained laboratory personnel following standardized procedures. This Direction For Use must be followed strictly. PAGE 2/6
B. Reagents and Materials Kylt MDV & Rispens DIVA Real-Time PCR Detection reagents contains the following reagents: Quantity in kit with Reagent Color code of lid 25 / 100 reactions Storage conditions 2.5x Detection-Mix violet 1 x / 4 x 500 µl -18 C to -20 C Positive Control red 1 x / 2 x lyophilisate (final 20 µl each) Negative Control (Nuclease-free water) +2 C to +8 C lyophilized -18 C to -20 C rehydrated blue 1 x 1 ml +2 C to +8 C All reagents are stored at 18 C to -20 C. Detection-Mix has to be stored dark, do not expose to direct (sun)light! The reagents are to be used within the indicated shelf life. Before the first use the lyophilized Positive Control is rehydrated: 20 μl of the Negative Control (Nuclease-free water) are added per vial of Positive Control, briefly incubated at room temperature and mixed thoroughly by repeated vortexing. Storage of aliquots with 5-10 μl volume (depending on the expected number of Positive Control reactions per kit) at -18 C to -20 C is recommended. The 2.5x MDV & Rispens DIVA Detection-Mix needs to be stored protected from light at -18 C to -20 C. Do not expose to direct (sun) light. Before first use, rehydrate the lyophilized 2.5x MDV & Rispens DIVA Detection-Mix: add 230 μl of the Negative Control per vial of 2.5x MDV & Rispens DIVA Detection-Mix, briefly incubate at room temperature and mix by pulse-vortexing. Generate aliquots of suitable volumes and store them at -18 C to -20 C. This kit can be used on all commercially available Real-Time PCR thermal cyclers that detect the emitted fluorescence of the fluorescent dyes FAM (emission 520 nm), HEX (emission 550 nm), Cy5 (emission 670nm) and Texas Red (620nm). We recommend to exclusively using certified Nuclease-free materials and powder-free protective gloves. Pipette tips have to be changed between samples to avoid cross-contamination. Gloves have to be changed frequently, especially after spillage or other suspected contaminations. Apart from the disposables, the following devices are needed (not included in this kit): DNA purification kit (e.g. Kylt RNA/DNA purification kit) Table top microcentrifuge Dry heating block (+100 C ± 3 C) Vortex Magnetic stirrer Micropipettes volume range 1-1000 µl Centrifuge for PCR tubes or plates Real-Time PCR thermal cycler PAGE 3/6
C. Control Reactions The Positive Control included in this kit allows for control of the specificity and efficiency of the reagents and the reaction itself. The Negative Control included in the kit allows exclusion of contaminations. The test is only valid if both, Positive and Negative Controls, are used and verified for validity in every Real-Time PCR run. An Internal Amplification Control is included in the Reaction-Mix in defined copy number; it is co-amplified (channel HEX) in every single reaction to detect possible inhibitory effects of the DNA-Extract and to verify true negative results. D. Protocol The overall protocol of this MDV & Rispens DIVA-analysis consists of the following steps: sample preparation, DNA extraction, Real-Time PCR and final analysis. 1. Sample Preparation We recommend pooling of not more than five individual samples or samples of more than five individuals, respectively, per DNA preparation. Feathers or swabs are pooled in a sufficient volume of sterile buffer (e.g. Normal Saline or 0.1 x TE) and soaked for an adequate period of time. Then, the sample is washed out thoroughly by pulse-vortexing. Immediately transfer the entire washed out supernatant to a conical screw cap tube. Tissue samples are thoroughly homogenized and a suitable volume is utilized for DNA preparation using appropriate kits. 2. DNA Extraction DNA Extraction can be performed using the Kylt RNA/DNA purification kit (Art. No. 31314/31315). For detail information on the DNA preparation process, please refer to the Direction For Use or Standard Operating procedure of the respective kit or in-house method, respectively. 3. Real-Time PCR Setup and Amplification Detection-Mix and Negative Control should be vortexed and spun down before each use. Determine the number of needed PCR-reactions needed: number of samples + Positive Control and Negative Control. Prepare the reaction mix for each sample using the components listed below. It is strongly recommended to prepare a Master-Mix consisting of qpcr-mix (e.g. BCD 2x qpcr-mix) and 2.5 x MDV Detection-Mix. Component Volume (µl) per reaction Final concentration qpcr-mix (e.g. BCD 2x qpcr-mix) 10 µl 1x 2.5 x MDV Detection-Mix 8 µl 1x Total Master Mix 18 µl - Template (Negative Control/sample DNA preparation/ Positive Control) 2 µl Total per reaction 20 µl PAGE 4/6
When calculating volumes for preparation the Master-Mix consider the respective concentration of the qpcr-mix used. For more information about the qpcr-mix please refer to the Direction For Use of the respective manufacturer. Add 18 μl of the Master-Mix to each of the PCR tubes or plate wells ( cavities ). Add 2 μl of Negative Control to corresponding cavity and seal. Add 2 μl of sample DNA-Extract to corresponding cavity and seal. Once all sample cavities are sealed, 2 μl of the Positive Control are added to the corresponding cavity and sealed. The Positive Control is vortexed and spun down before each use. Avoid formation of bubbles when pipetting Reaction-Mix, samples and controls. It is recommended to always centrifuge cavities before the PCR run. Place the cavities in the Real-Time PCR thermal cycler and amplify using the following parameters: Step Temperature Duration Activation Polymerase 95 C 10 min Denaturation 95 C 15 sec Annealing & Extension 60 C 1 min Fluorescence Detection channels FAM, Cy5, Texas Red and HEX 42 cycles Please follow the specific instructions of your Real-Time PCR thermal cycler as recommended by the manufacturer. Depending on the qpcr-mix used and its respective temperature profile this Kylt MDV & Rispens DIVA Real-Time PCR Detection Reagents allow the combination with other Kylt Real-Time PCRs for detection of bacteria. When combining Kylt detection tests for different pathogens, make sure all necessary channels are used! 4. Analysis General The data of the amplification reactions can be automatically processed using specific software for the Real-Time PCR thermal cycler. Alternatively a threshold can be set manually. The threshold should cross the HEX-curve of the Negative Control and the FAM-, Cy5- and Texas Red -curve of the Positive Control, respectively, in the linear area of its slope. By setting the thresholds, the crossing points with the HEX- and FAM, CY5- and Texas Red-curves determine the respective cycle threshold (Ct), which is negatively correlated with the initial concentration of copies of the target genes in the Real-Time PCR reaction. Only curves with the typical exponential amplification, meaning the curve of the raw data shows a fl at baseline at the beginning, followed by a clear (exponential) slope in fluorescence and possibly reaching a plateau-phase (y-axis in logarithmic scaling), should be regarded as positive. For the test analysis the validity of the run is determined with the Negative and Positive Controls. After that the validity of each single sample is verified by Internal Amplification Control reaction and finally their MDV & Rispens DIVA-specific status is checked. Test Evaluation The test is only valid if the FAM, CY5- and Texas Red-curves of the Negative Control is negative, if the HEX-curve of the Negative Control is positive and if the FAM-, CY5- and Texas Red-curves of the Positive Control is positive. PAGE 5/6
HEX-curve positive yes yes / no yes / no no FAM-curve positive no yes yes no Cy5-curve positive no yes no no Texas Red-curve positive no no yes no The sample is MDV negative positive positive inhibited The sample is MDV Rispens negative positive negative inhibited The sample is MDV field strain negative negative positive inhibited The sample is positive for Marek Disease Virus if the FAM channel is positive, independent of the channels Cy5 and Texas Red. A sample is positive for the Marek Disease virus vaccine strain Rispens CVI988 if the sample is positive in the FAM- and Cy5- channel. A sample is positive for the Marek Disease virus field strain if the sample is positive in the FAM- and Texas Redchannel. A potential double infection with Marek Disease Virus field strain and the Rispens vaccine strain is possible if the FAM-, Cy5- and Texas Red-curves are positive. A sample is inhibited if neither the FAM, Cy5- and Texas Red-curves nor the HEX-curve are positive. Recommendation: In the case of an inhibited sample the test should be repeated with a dilution of the DNA extract at 1:10 (9 volumes Negative Control + 1 volume DNA extract or eluted DNA). The Negative Control serves as the diluting agent. Convenient and reliable sample data entry, Real-Time PCR start, final qualitative analysis and documentation can be conducted with the Kylt PCR Software package, please inquire. Production: AniCon Labor GmbH Muehlenstr. 13a D-49685 Hoeltinghausen Germany www.anicon.eu www.kylt.eu Kylt is a registered trademark. For veterinary use only. For in vitro use only. Regulatory requirements vary by country, this product may not be available in your geographic area. 2017 AniCon Labor GmbH. All rights reserved. The trademark mentioned herein is the property of AniCon Labor GmbH or their respective owners. THE PURCHASE OF THIS PRODUCT GRANTS THE PURCHASER RIGHTS UNDER CERTAIN ROCHE PATENTS TO USE IT FOR PROVIDING VETERINARY IN VITRO DIAGNOSTIC TESTING. NO GENERAL PATENT OR OTHER LICENSE OF ANY KIND OTHER THAN THIS SPECIFIC RIGHT OF USE FROM PURCHASE IS GRANTED HEREBY. PAGE 6/6 q.dr.mdv DIVA.02 Rev001, July 2017